Scientific Core 2:Mass Spectrometry and Biophysics

科学核心2(SC2)。项目摘要/摘要 质谱学和生物物理方法在本计划项目的各个方面的应用 这项研究计划的一个独特方面。科学核心2(SC2)的目的是提供两个质量 为计划项目中的研究人员提供光谱和生物物理支持。的主要目标 SC2将提供强大的、最先进的技术，用于准确和可重复地测量 大分子相互作用与蛋白质物理性质。为了实现这一目标，核心服务于 所有三个项目在研究蛋白质特性、构象变化和 蛋白质之间的相互作用。第二个主要目标是质谱学检测和量化 小分子，包括生物活性的氧化脂质和氧化氨基酸，它们是氧化的报告者 流程。SC2是分析、结构和建模/计算方面的专业知识的集中领域 生物物理学。核心不仅提供对仪器和试剂的访问，更重要的是提供 在建立方法、分析和解释结果以及确保质量方面的经验和专业知识 控制力。核心由两个主要的子组成部分组成，它们的方法不同，但互补性很强 方法：一个质谱子部分和一个生物物理子部分。SC2将提供 通过表面等离子体共振光谱分析检测直接相互作用的专业知识和试剂 报告了结合的开启和关闭速率以及解离常数Kd的准确测定。这 方法由等温滴定量热法补充用于热力学表征 大分子组装。SC2的质谱子组件将有助于识别 氢-氢交换-质谱仪(HDX-MS)测定蛋白质-蛋白质相互作用位点， 这是一个强大的工具，也提供了关于蛋白质动力学的信息。这一方法还有两个补充 生物物理子组件提供的方法-Förster共振能量转移和系留 人工蛋白酶法(“Fe-Babe”)。核心提供蛋白质和它们的模型的专业知识 使用已知结构、蛋白质-蛋白质对接方法和分子动力学模拟的相互作用。 质谱学为蛋白质组学分析提供了一种强大的发现工具，有助于蛋白质的鉴定 以及翻译后的修改。另一种质谱学仪器将用于 小分子的检测和定量，包括生物活性氧化脂质和修饰氨基酸 这些都是氧化应激的特定记者。公务员事务局局长会提供多项服务，包括 试剂、探头设计和建造、实验设计和性能以及数据方面的协助 对结果进行分析和解释。教育部分是这一核心的额外优势。全 SC2提供的方法需要高度专业化的仪器、技术专长和经验说明 通常在现代细胞或分子生物学实验室中可用；此外，一些方法需要 不熟悉的计算分析方法。因此，质谱学和生物物理学核心提供了关键的 对计划项目的成功至关重要的能力。

Scientific Core 2 (SC2). Project Summary/Abstract The application of mass spectrometry and biophysical approaches to various facets of this Program Project is a unique aspect of this research program. The purpose of Scientific Core 2 (SC2) is to provide both mass spectrometric and biophysical support for the investigators within the Program Project. A principal objective of SC2 is to make available robust, state-of-the-art techniques for accurate and reproducible measurement of macromolecular interactions and protein physical properties. Addressing this objective, the Core serves the common need of all three Projects for investigation of protein characteristics, conformational changes, and protein-protein interactions. A second principal objective is mass spectrometric detection and quantification of small molecules, including bioactive, oxidized lipids and oxidized amino acids that are reporters of oxidative processes. SC2 serves as a centralized area of expertise in analytical, structural, and modeling/computational biophysics. The Core provides not only access to instrumentation and reagents, but more importantly provides experience and expertise in setting up the methods, analyzing and interpreting the results, and assuring quality control. The Core consists of two major sub-components with different but highly complementary methods and approaches: a Mass Spectrometry Sub-component and a Biophysics Sub-component. SC2 will provide expertise and reagents for detection of direct interaction by surface plasmon resonance spectrometry that reports accurate determinations of on- and off-rates of binding, as well as dissociation constants, KD. This method is complemented by isothermal titration calorimetry for thermodynamic characterization of macromolecular assemblies. The Mass Spectrometry Sub-component of SC2 will facilitate identification of protein-protein interaction sites by hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), a powerful tool that also provides information on protein dynamics. This approach is supplemented by two methods provided by the Biophysics Sub-component - Förster resonance energy transfer and the tethered artificial protease procedure (“Fe-BABE”). The Core provides expertise in modeling of proteins and their interactions using known structures, protein-protein docking methods, and molecular dynamics simulations. Mass spectrometry provides a powerful discovery tool in proteomic analysis useful in identification of proteins as well as post-translational modifications. Alternative mass spectrometry instrumentation will be used for detection and quantification of small molecules, including bioactive oxidized lipids and modified amino acids that are specific reporters of oxidative stress. SC2 will provide a diversity of services including provision of reagents, assistance in probe design and construction, design and performance of experiments, and data analysis and interpretation of results. An educational component is an additional strength of this Core. All methods provided by SC2 require highly specialized instrumentation, technical expertise, and experience not generally available in a modern cell or molecular biology laboratory; additionally, some methods require unfamiliar computational analysis methods. Thus, the Mass Spectrometry and Biophysics Core provides critical capabilities essential for the success of the Program Project.